The plastids of higher plants are an attractive target for genetic engineering. Plant plastids (chloroplasts, amyloplasts, elaioplasts, etioplasts, chromoplasts, etc.) are the major biosynthetic centers that, in addition to photosynthesis, are responsible for production of industrially important compounds such as amino acids, complex carbohydrates, fatty acids, and pigments. Plastids are derived from a common precursor known as a proplastid and thus the plastids present in a given plant species all have the same genetic content. In general, plant cells contain 500-10,000 copies of a small 120-160 kilobase circular genome, each molecule of which has a large (approximately 25 kb) inverted repeat. Thus, it is possible to engineer plant cells to contain up to 20,000 copies of a particular gene of interest which potentially can result in very high levels of foreign gene expression. In addition, plastids of most plants are maternally inherited. Consequently, unlike heterologous genes expressed in the nucleus, heterologous genes expressed in plastids are not pollen disseminated, therefore, a trait introduced into a plant plastid will not be transmitted to wild-type relatives.
Plastids of higher plants present an attractive target for genetic engineering. As mentioned above, plastids of higher plants are maternally inherited. This offers an advantage for genetic engineering of plants for tolerance or resistance to natural or chemical conditions, such as herbicide tolerance, as these traits will not be transmitted to wild-type relatives. A review of plastid transformation of flowering plants is provided by Maliga (1993) Trends in Biotech. 11:101-107, the entirety of which is incorporated herein by reference.
Unfortunately, successful plastid transformation techniques described thusfar for higher plants have been limited to model crop plants such as tobacco (U.S. Pat. No. 5,451,513; Svab et.al. (1990), Proc. Natl. Acad. Sci. USA 87:8526-8530 and Svab et al. (1993), Proc. Natl. Acad. Sci. USA 90:913-197) and Arabidopsis (Sikdar, et al. (1998) Plant Cell Reports 18:20-24). Furthermore, the methods described for Arabidopsis plants, produce infertile regenerates. PCT Publication WO 97/32977 also describes methods for the plastid transformation of Arabidopsis and provides prophetic examples of plastid transformation of Brassica plastids. However, transplastomic Brassica plants have not been produced to date using the methods described therein. Thus, for practical applications of genetic engineering techniques to crop plant plastids, chloroplast transformation techniques for a wide variety of crop plants, such as Brassica species, are needed in the art.
Chloroplast transformation methods applicable to crop species, such as Brassica species, are needed in the art. Such methods would provide for a novel means of genetic engineering via plastid transformation for agronomically as well as qualitatively important traits via genetic engineering of plant plastids.